DNA Methylation Application Arrays
DNA Methylation Application Arrays
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Note: This is a review of the published article listed below. All information, quotes, figures, methods, and findings mentioned in this review are from that article, and are the property of its authors and/or the publication in which the article originally appeared.
Methylation of CpG dinucleotides in genomic DNA, a primary epigenetic modification in humans, is known to be closely correlated with cellular processes such as cancer, gene silencing, and genomic imprinting. The DNA methylation microarrays offered by Agilent are designed to help you identify and characterize these important regulatory events. Featuring in situ synthesized 60-mer oligonucleotide probes and a two-color labeling system, our line of CpG island, promoter, and whole genome tiling arrays provide sensitive and specific comprehensive DNA methylation profiling.
The application of Agilent Human CpG island microarrays in the identification of aberrantly methylated genes in cancer cells and tissue was recently reported at the 2008 American Association for Cancer Research (AACR) meeting. The Human CpG island microarray, developed to represent the majority of CpG islands in the human genome, contains ~200,000 oligonucleotide probes tiling the 21 megabases of ~25,000 unique CpG islands, with an average spacing of 95 base pairs. The microarray is designed to be compatible with several published methods for the genome-wide detection of methylated CpG islands.
In this work, Agilent researchers and colleagues at UC Davis and Hebrew University used methylated DNA immunoprecipitation (mDIP) in conjunction with Human CpG Island microarrays to obtain comprehensive methylation profiles of normal human liver DNA. A schematic of this workflow is shown in Figure 1. Bisulfite sequencing was used to confirm the methylation patterns generated by the microarrays (Figure 2). The validated assay was then used to identify abnormally methylated genes in lung cancer cells and colon carcinoma tissue. DNA methylation profiling of the human lung cancer NCI-H69 cell line showed high levels of methylation in the promoter regions of the HoxA gene cluster (Figure 3). Comprehensive analysis of CpG islands in colon carcinoma tissue identified four putative colon cancer methylation markers (Figure 4).
Figure 1. Schematic of the experimental workflow.
Genomic DNA is randomly sheared to an average length of 400bp by sonication. The sheared genomic DNA is immunoprecipitated with a monoclonal antibody against 5-methylcytosine to isolate and enrich methylated DNA from the genome. The resulting fraction of methylated DNA is purified, cyanine 5 (Cy-5)-labeled, and competitively hybridized against cyanine 3 (Cy-3)-labeled “input” genomic DNA onto a single microarray. The microarray is washed, scanned and analyzed with Agilent DNA Analytics software. Relative DNA methylation levels for each probe/CpG island are reflected in changes in Cy-5/Cy-3 ratios.
Figure 2. Plot of probe ratios and ROC curves for a selection of probes within CpG islands with bisulfite sequencing confirmed methylation patterns.
(A) MDIP was performed on normal adult liver DNA. Bisulfite sequence data across ~65 CpG islands in normal liver DNA was obtained from the Human Epigenome Project and from in-house efforts. (A) Plot of binned log ratios of all probes by count. Note the bimodal distribution of signals which can aid in defining a cutoff for methylated probes. (B) Plot of Ratio by probe TM colored by bisulfite methylation status. Probes corresponding to islands with >90% methylation by bisulfite sequencing are classified as methylated (red points) and islands with less than 10% methylation are classified as unmethylated (blue points). A line was fit to maximize separation of probe ratios from methylated and unmethylated regions. (C) Receiver Operator Characteristic curve fit of the false positive rate vs. the true positive rate.
Figure 3. Comparison of methylation profiles of lung cancer and normal lung cell lines at the HoxA gene cluster on Chromosome 7.
Microarray analysis of methylated DNA IPs for NCI-H69 small cell lung carcinoma (A; orange line) and HEL299 normal embryonic lung fibroblast (B; blue line) genomic DNA. For each sample Cy5-labeled, meDIP enriched DNA and Cy3-labeled input genomic DNA were competitively hybridized to an Agilent Human CpG Island Microarray (P/N G4492AA). Microarrays were scanned on an Agilent Scanner and data normalized to a median log2 ratio of zero. Each point represents an individual probe on the microarray. Probes with enrichment levels above the median correspond to higher methylation levels and are colored red. Probes corresponding to regions of less enrichment/methylation are colored green. The orange line (A), or the blue line (B), represent a three-point moving average of the log2 ratio. Statistically significant hypermethylated (regions with log ratios significantly above zero) or hypomethylated (regions with log ratios significantly below zero) are further elucidated with shading and horizontal bars. The promoter regions of HoxA7 and HoxA9 display high levels of relative methylation in NCI-H69 (A). These same regions show much lower levels of relative methylation in the normal lung fibroblasts (B). Similar findings were recently reported in primary lung (Rauch et al., 2007) and in breast cancer (Novak et al., 2006).
Figure 4. Comparison of methylation profiles of colon cancer and normal colon tissue at several promoter regions.
Microarray analysis of methylated DNA IPs for DNA from three normal colon and three colon carcinoma tissue samples. Red lines denote a 3 probe moving average across each displayed island for Carcinoma. Blue lines represent a three point moving avg across each displayed region for normal tissue. Green lines represent CpG islands. MDIP enriched DNA and Cy3-labeled input genomic DNA were competitively hybridized to an Agilent Human CpG Island Microarray (P/N G4492A). Microarrays were scanned on an Agilent Scanner and data normalized to a median log2 ratio of zero. Each point represents an individual probe on the microarray. Probes with enrichment levels above the median correspond to higher methylation levels and are colored red. Probes corresponding to regions of less enrichment/methylation are colored green. The red lines represent a three-point moving average of the log2 ratio. (Shen et al., 2007, Sato et al., 2007).
Original Poster
Title: Genome-wide Microarray Analysis of CpG Island Methylation.
Authors: Douglas Roberts, Alex Wong, Shane Giles, **Ravid Straussman, *Jeff Gregg, *Ryan Davis, *Brian Poirier, Bo Curry, Zohar Yakhini, Jayati Ghosh,*Howard Cedar, Douglas Amorese
Agilent Technologies, 5301 Stevens Creek Blvd., Santa Clara, CA 95051, *Dept. of Pathology, UC Davis Medical Center, Sacramento, CA, and
**Dept. of Cellular Biochemistry and Human Genetics, Hebrew University, Jerusalem, Israel
To download the full poster click here.
Human CpG Island: Human CpG Island: gain insights into biological processes such as cancer, development, genomic imprinting, gene silencing, and chromatin stability using a powerful 244K array analysis tool for genome-wide measurements
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Human 2-Set Promoter: the promoter array is designed for location analysis of human DNA-binding proteins and targets approximately 17,000 of the best-defined human transcripts on a 244K array
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Mouse CpG Island: a powerful 2X105K array analysis tool for genome-wide measurements in development, genomic imprinting, gene silencing, and chromatin
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Mouse Promoter: designed for location analysis of mouse DNA-binding proteins, targeting approximately 17,000 of the best-defined mouse transcripts on a 244K array
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Human Whole Genome: a broad view that represents all known genes and transcripts in the human genome
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Mouse Whole Genome: a broad view of the entire mouse genome that represents all known genes and transcripts
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Arabidopsis Whole Genome: explore gene regulation in embryonic and adult development as well as signaling networks using probes tiled across the entire Arabidopsis genome
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